Early in this study, an α-Amylase from Bacillus megaterium WHO (BMW) was isolated from hot springs of Ramsar (North of Iran), and its gene was cloned in E.coli. Based on its conserved sequence regions and substrate specificity, it was classified as intermediary group enzymes with the specificity of oligo-1,6-glucosidase and neopullulanase subfamilies. In the sixth conserved region (83-QVNGIWMMP), like oligo-1,6-glucosidase subfamily, there is a highly conserved Trp, instead of Tyr for neopullulanase subfamily. In this study, through Trp88Tyr mutation the role of this amino acid in the substrate specificity of enzyme was investigated. The specificity of enzyme against starch, pullulan, amylose and amylopectin was determined. Compared to the wild type, thermal stability and the catalytic efficiency of the mutant increased while product pattern of enzyme didn’t change by mutation. As expected, the neopullulanas activity of enzyme increased.